We plan to investigate the endocrine mechanisms involved in the control of melanogenesis and growth of cultured melanoma cells. Because continued transcription and translation is necessary for MSH action, studies will be carried out to determine if cAMP stimulates tyrosinase activity by causing de novo enzyme synthesis. Immunoprecipitation techniques will be utilized in these studies to quantitate the amount of tyrosinase synthesized in either control or MSH-treated cultures. Further, the level of functional mRNA coding for tyrosinase will be determined with the use of an in vitro translation system derived from wheat germ. The nuclear role in mediating the hormonal induction of tyrosinase will also be investigated by: 1) examining the effects of either MSH or cAMP on tyrosinase activity in enucleated cell cultures, and 2) determining hormonal effects on RNA polymerases I, II, and III. Because cAMP might stimulate melanogenesis in melanoma cells by not only increasing tyrosinase synthesis but also by enhancing the activity of the enzyme, the effect of cAMP dependent protein kinase on the phosphorylation and activity of purified tyrosinase will be studied. The phosphorylation of melanosomes by protein kinase and the effect of this event on melanosomal bound tyrosinase activity will be assessed. Studies will also be carried on to investigate the possibility that the two enzymatic activities of tyrosinase can be uncoupled in either purified enzyme or in situ. We will continue our investigation of the possible modulatory effects of steroid hormones, insulin, retinoids, and thyroid hormones on the cAMP regulation of tyrosinase activity. Finally, we will determine the phase of the cell cycle at which cells are blocked by cAMP.